外周血白细胞凋亡障碍与大鼠肠缺血-再灌注损伤的关系

目的： 探讨外周血白细胞和中性粒细胞凋亡障碍在肠缺血-再灌注（IR）损伤中的作用。方法：20只雄性SD大鼠随机分为肠IR组和假手术对照组，每组10只。肠IR组夹闭肠系膜上动脉（SMA）30 min后再灌注60 min；对照组只分离而不夹闭SMA。观察肠黏膜形态学变化；检测肠黏膜上皮细胞凋亡指数（TUNEL法）和caspase-3活性；Annexin-V/PI法检测外周血白细胞和中性粒细胞（PMN）凋亡比例；检测夹闭前、夹闭30 min、再灌注30 min、再灌注60 min外周血白细胞数量。结果：(1)光镜下，对照组未见肠黏膜损伤，而IR组肠黏膜损伤严重；（2）IR组肠黏膜上皮细胞凋亡指数和caspase-3活性比对照组明显增高（P<0.05）；（3）IR组外周血白细胞和PMN凋亡比例比对照组明显降低（P<0.05）；IR组外周血白细胞数量在缺血30min时明显增高，再灌注后进一步增高，与缺血前和对照组比都有显著差异（P<0.05）。(4)肠黏膜caspase-3活性与外周血白细胞凋亡比例有显著的负相关（r=-0.764, P<0.01），与PMN凋亡比例也有显著的负相关（r=-0.845，P<0.01）；肠黏膜上皮细胞凋亡指数与PMN凋亡比例也有显著的负相关（r=-0.638, P<0.05）。结论：外周血白细胞数量增加及凋亡障碍与缺血-再灌注引起的肠黏膜细胞损伤密切相关。

Correlation of peripheral leukocyte apoptosis insufficiency and intestinal injury following mesenteric ischemia/reperfusion in rats

AIM: To investigate the role of peripheral blood leukocytes and polymorphonuclear neutrophils (PMNs) on intestinal injury following mesenteric ischemia/reperfusion (IR) in rats. METHODS: Twenty adult, male Sprague-Dawley rats, weighing 200-230 g, were randomly divided into two groups. The control group (CON) consisting of 10 rats was subjected to laparotomy and separation of superior mesenteric artery (SMA) only. The ischemia/reperfusion (IR) group consisting of 10 rats, was subjected to laparotomy, followed by occlusion of the superior mesenteric artery (SMA) by an atraumatic microvascular clamp for 30 min. At the end of ischemic period in IR, the microvascular clamp was removed and the intestinal segment was reperfused for 60 min. The pathological changes of the ileal mucosal tissue were evaluated. The apoptosis of intestinal mucosal epithelial cells was examined by terminal deoxylnucleotidy-l transferase mediated-dUTP nick end-labeling (TUNEL). The enzymatic activity of casapse-3 in mucosal cells was determined using a colorimetric assay. The percentages of apoptotic peripheral blood leukocytes and PMNs were measured by flow cytometry using Annexin-V/PI double staining assay. The numbers of peripheral blood leukocytes in each animal was measured at baseline, 30 min of ischemia, and 30 min and 60 min of reperfusion. RESULTS: (1) Compared to CON group animals, the most severe mucosal injury was observed in IR group under optical microscope. (2) The number of apoptotic mucosal epithelia cells and enzymatic activity of caspase-3 were significantly higher in IR than those in CON group (P<0.05). (3) The percentages of apoptotic peripheral blood leukocytes and PMNs were significantly lower in IR group than those in CON group (P<0.05). The number of peripheral blood leukocytes in IR group was increased obviously after SMA closed for 30 min (P<0.05), and was higher following reperfusion (P<0.05), and significantly higher than that in CON group (P<0.05). (4) A negative correlation between the enzymatic activity of caspase-3 in the ileal mucosal tissue and the percentage of apoptotic peripheral blood leukocytes (r=-0.764, P<0.05), and of PMNs (r=-0.845, P<0.05) was found. A negative correlation between the apoptosis index of mucosal epithelial cells and the apoptotic percentage of PMNs (r=-0.638, P<0.05) was also observed. CONCLUSION: Apoptosis insufficiency and higher numbers of peripheral blood leukocytes are correlated with the mucosal cells injury following mesenteric ischemia/reperfusion (IR) in rats.